The goal of the proposed studies is to elucidate, using patch clamp techniques, the mechanisms by which chronic exposure to mu-opioid agonists results in a tolerant state in cultured rat neonatal dorsal root ganglion (DRG) neurons. To do this, the signaling pathways responsible for mediating the acute actions of opioids will be investigated in order to test the hypothesis that the tolerant state results from altered receptor-G protein interactions. Mu- and kappa- Opioid agonists decrease or increase voltage-dependent Ca 2+ currents in DRG neurons: inhibitory responses are mediated by Go-type G proteins, but of unknown type; excitatory responses are not as well described. DRG neurons exposed to the Mu agonist DAMGO for greater than 2 days become tolerant to the acute inhibitory actions of Mu agonists and exhibit excitatory responses more frequently. The first phase of the proposed work will test the hypothesis that Mu and Kappa agonists affect the same Ca 2+ channel subtypes, with inhibitory responses mediated by the same Go subtype. The targeted channel subtypes will be identified using specific blockers of Lll-, N-, P- and Q-type channels. The G proteins will be identified by blocking opioid responses by intracellular perfusion with G-protein-specific antibodies; by transfection of antisense oligonucleotides to distinct G protein alpha subunits; and by intracellular perfusion of G proteins or subunits to restore opioid responses in neurons treated with pertussis toxin. The next phase will test the hypothesis that the acute inhibition of Ca 2+ channels by opioids is "tightly coupled" but is influenced by second messenger pathways. First, opioid agonists will be applied externally during an on-cell recording. Next, intracellular perfusion of protein kinases A or C, their inhibitors, or phosphatase inhibitors will determine the role of these pathways in mediating or supporting opioid actions. The third phase, using the above methods, will test the hypothesis that excitatory responses target L-type channels via Gs in a membrane-delimited fashion. The final phase will test, again using the above approaches, the hypothesis that the development of a tolerant state in vitro results from a change in opioid receptor-G protein interactions; the role of kinases will be determined as well. The results of these studies will reveal novel information about opioid signaling pathways in the non-tolerant and tolerant states and thus will support further investigations of opioid action and the development of better analgesics.